Project Summary Recent efforts have focused on developing T cell-based immune therapies to treat cancer. However, these therapies have several limitations, including minimal efficacy against solid tumors. One characteristic of the solid tumor microenvironment is low oxygen availability, or hypoxia. It is generally thought that tumor hypoxia suppresses the anti-tumor immune response. The goal of this project is to improve our understanding of how T cells respond to hypoxia and reveal new strategies for increasing the anti-tumor function of hypoxic T cells. These findings will ultimately inform the engineering of T cell therapies to overcome the hypoxic tumor microenvironment. Two key T cell subtypes that modulate anti-tumor immune function are pro-inflammatory T effector (Teff) and anti-inflammatory T regulatory (Treg) cells. Previous studies examining the effects of hypoxia on these cell types are highly discordant. Clarifying the effects of hypoxia on Teff and Treg function will expand our understanding of basic T cell biology and potentially identify new means of enhancing anti-tumor T cell activity in hypoxia. Here, we hypothesize that hypoxia alters T cell surface proteins (the ?surfaceome?) and T cell function in a manner consistent with a net immunosuppressive effect. To test this hypothesis, we will utilize a combination of studies with primary human T cells, animal models, and patient samples. We will employ our established surfaceomic and phage display-based recombinant antibody engineering strategies to examine the effect of hypoxia on suspected hypoxia-induced antigens (HIAs) as well as to identify novel HIAs. We will first apply these techniques to isolated primary human Teff and Treg cells cultured in normoxic or hypoxic conditions in vitro. We will then examine hypoxic tumor-infiltrating T cells derived from humanized mouse models of pancreatic cancer as well as patient pancreatic tumor samples. This surfaceomic profiling, combined with T cell proliferation, activation, and migration studies, will provide a comprehensive picture of hypoxia-induced T cell surface protein and functional changes. Furthermore, we will engineer novel inhibitory antibodies or bispecific constructs targeting both suspected and newly-identified HIAs. These engineered proteins will be tested for Teff/Treg modulatory function in hypoxic culture and in a humanized pancreatic cancer mouse model with the ultimate goal of identifying strategies to promote hypoxic Teff function and immune-mediated tumor killing. In the long term, the basic biological insights and antibody tools gained from the proposed studies will inform ongoing efforts to treat cancer using T cells and will lay the foundation for future therapeutics to boost immune targeting of hypoxic tumors.